The impressive achievements in genomic sequencing over the past several years have yielded a wealth of information for many model organisms, including the model plant Arabidopsis thaliana. At the same time, the knowledge that sequence information alone is insufficient to answer many biological questions concerning gene function, development, regulation, and biochemical kinetics have defined the need for more comprehensive approaches. To address these questions, we are employing an integrative approach for the analyses of gene expression products at the transcriptome, proteome, and even metabolome levels. Transcriptome approaches using microarray and SAGE technologies are powerful tools, however, mRNA abundances may only represent putative function since there is still a questionable correlation between mRNA and protein levels (Futcher et al., 1999; Gygi et al., 1999) . In contrast, proteomics provides a more direct approach to biochemical processes by profiling the actual protein constituents performing the enzymatic, regulatory, and structural functions encoded by the genome and transcriptome at a given point in time.

Recent improvements in high resolution, two-dimensional polyacrylamide gel electrophoresis (2-DE) (Gorg et al., 1999; Klose and Kobalz, 1995) , increased content of protein and nucleotide databases, and the increased capabilities for protein identification utilizing modern mass spectrometry methods such as MALDI-TOF/MS (Pappin et al., 1993; Rabilloud, 2000; Yates III, 1998; Yates, 1998) , has made the large scale identification of proteins a dynamic new area of research in plant biology. Accordingly, we are utilizing 2-DE to probe the proteome of Medicago truncatula.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been established as the dominant technique for analysis of complex protein mixtures since its introduction in 1975.23,24 The technique utilizes isoelectric focusing and polyacrylamide gel electrophoresis for first and second dimension separation, respectively. Currently, 2-D PAGE technology is capable of resolving some 10,000 proteins, with 2,000 proteins being typical experimental results.25 A recent review describes the role of 2-D PAGE in proteomic and genetic studies of plant systems, including its use as a tool to investigate genetic diversity, phylogenetic relationships, mutant characterization, and drought tolerance.26 2-D PAGE has also been utilized in studies on plant defense-associated responses and responses to methyl jasmonate.25-28

2-DE profile of Medicago truncatula suspension cell culture protein extract.

Although 2-D PAGE analysis has been used for the last 20 years in protein profiling, it provides limited information on protein identification. Recent advances in mass spectrometry and the establishment of protein databases have substantially increased the ease and speed with which proteins can be identified.29 The union of these technologies is the foundation for modern proteomic studies. The typical experiment begins with comparative digital imaging of the 2-D gels to detect variations in protein concentration or elution profile. These protein spots are excised, extracted, and identified by using a variety of mass spectrometry techniques.

Basically, two mass spectrometry (MS) techniques are used for protein identification. The first is peptide mass-mapping of proteolytic digest fragments.29,30 The observed mass fragments can be searched against a theoretical list of proteolytic peptide maps predicted by a given database. Increased peptide mass accuracy has increased the success and selectivity of such searches.31 If the database query is unsuccessful, the protein can be sequenced by using tandem mass spectrometry (MS/MS).29 During the MS/MS experiment, only the peptide mass of interest is isolated or transmitted, thus discriminating against all other components of the mixture with different mass values. After isolation, the peptide is further fragmented by using a unimolecular or bimolecular (collision gas) strategy. Fragments observed in the isolated peptide MS/MS spectrum can then be rationalized to a sequence.

Initial proteome profiling in our program has been performed to generate representative 2-D PAGE protein profiles for stems, leaves, seedpods, roots, flowers, tissues, and suspension cell cultures. Proteins were systematically identified and cataloged by using peptide mass mapping and database searching. An interactive database of the results of these analyses can be found at the following Web address: /Website/2DPAGE/search.asp. Analytical and biological variances associated with the 2-D PAGE proteomics approach for M. truncatula have been determined and will function as baseline measurements for comparative protein profiling in elicitor-induced M. truncatula cell cultures.

See Also:

The Sumner Group: Bioanalytical Chemistry and Mass Spectrometry

References